Morphometric enumeration of lymphocyte subsets in the gut-associated tissues of SIV infected and uninfected macaques. Histological examination of the small and large intestine of five monkeys infected intracolonically with SIV/DeltaB670 was performed on biopsied tissue obtained 3 weeks postinoculation and compared to tissues from 2 healthy naive animals to identify early changes in gut-associated tissues induced by virus infection. Computer-assisted morphometric evaluation was performed on stained tissues to allow in situ quantitation of cells expressing various markers. Immunolocalization of nitrotyrosine was used in this study to localize the presence of peroxynitrite (an indicator of nitric oxide activity) in tissues. Elevated numbers of nitrotyrosine expressing cells were present in the lamina propria of both the colon and ileum of infected animals. The cell types staining positive for nitrotyrosine appeared to be macrophages. These results are not surprising given that macrophages can be activated by cytokines produced by sensitized lymphocytes responding to specific viral antigens (such as SIV). By 3 weeks postinfection with SIV/DeltaB670, elevated numbers of cells in the lamina propria were found to express TNF`, with the colon containing a greater number of cells than the ileum. These findings are consistent with the previously observed elevated levels of serum TNF` associated with systemic SIV infection. Both TNF` and peroxynitrite formation appear to occur simultaneously in the intestine following SIV inoculation. Taken together, these findings clearly identify early changes associated with mucosal exposure to pathogenic SIV that are diagnostic for infection and suggest possible avenues for identification of changes which either predispose to, or are associated with, the induction of protective responses associated with transient infections. As such, this technology will prove highly useful in analysis of the mucosal tissues obtained from the monkeys described in this project. Indeed, a preliminary survey of nitric oxide positive cells in intestinal biopsies obtained from a group of 4 animals colonically inoculated with the immunizing virus, SIV/17E-Fr, 2 of which had virus-specific CTL activity in the lamina propria and were subsequently protected upon colonic challenge, and 2 which did not is fascinating. Morphometric evaluation of NO producing cells in the ileal and colonic lamina propria prior to exposure with the immunizing virus, SIV/17E-Fr, was compared to that obtained 8 weeks later. There were clear differences between pre- and post-exposure tissues in all 4 monkeys; however, the levels observed in monkeys that had no evidence of CTL (and were subsequently not protected) were significantly higher than in those where CTL could be identified (These animals were shown to be protected upon subsequent challenge with SIV/DeltaB670). Virology. To date, we have inoculated 11 monkeys intracolonically with limiting doses of either SIVmac239 or its congenic macrophage-tropic derivative, SIV/17E-Fr. Inoculations were performed by endoscopic insertion of a cell-free virus preparation approximately 50 cm into the colon. Four monkeys received either 10 or 100 TCID50 of SIVmac239. A disseminated infection resulted in only one of these monkeys as evidenced by a) repetitive identification of SIV sequences by PCR; b) isolation of virus; and c) seroconversion. The remaining monkeys remained seronegative and virus culture negative, but virus sequences were sporadically identified in PBMC by PCR. Seven monkeys were similarly inoculated with 100 or 1000 TCID50 SIV/17E-Fr. Of these, 2 monkeys had clear signs of a disseminated infection; and, of the remaining monkeys, only one monkey had any other signs of virus exposure (PCR+ at one time point). Seven animals were challenged intracolonically with 10,000 TCID50 SIV/DeltaB670 6 months after exposure with the immunizing virus; whereas, the remaining 4 monkeys were challenged 3 months post-exposure. Four naive control animals were also challenged with this dose to validate the infectivity of the challenge inoculum. All 4 controls became infected as evidenced by PCR detectable sequences in PBMC, and developed disease at variable rates like that seen in intravenously inoculated animals. In contrast, three of the 11 previously exposed animals remained virus negative and seronegative following challenge. SIV gp120 V1 sequences of virus present in all PCR+ animals was amplified, cloned, and sequenced to confirm the presence or absence of the challenge virus (There is a 28% sequence divergence in V1 between the 2 clones and SIV/DeltaB670 so the immunizing and challenge viruses are readily discernible by this analysis). SIV/DeltaB670 sequences were identified in all PCR+ monkeys at 2 weeks postchallenge. Thus, of the 11 monkeys previously exposed to limiting doses of either SIVmac239 or SIV/17E-Fr, 4 monkeys appeared protected from high dose challenge with the primary isolate, SIV/DeltaB670. Three of these animals remained PCR negative following exposure with both the immunizing and challenge viruses, and one, had only a transient appearance of the challenge virus in the periphery postchallenge. These results were further validated by monitoring disease progression by longitudinal analysis of T cell subsets. All monkeys where SIV/DeltaB670 could be persistently identified showed declines in either CD4+ or CD4+CD29+ T lymphocytes and increases in CD8+T lymphocytes postchallenge; whereas the 4 monkeys that appeared to be protected remain immunologically unchanged.